Targeted mutations in B. pseudomallei and B. mallei have demonstrated a central role for type III secretion in virulence (134,150). Despite their obvious importance, the vast majority of effectors remain uncharacterized. Furthermore, although is it clear that TsSS-3 plays a major role in pathogenesis in rodents, TsSS-i and -2 are almost completely unexplored. Finally, the extent and consequences of diversity in the complement of effector loci present in different B. pseudomallei isolates has yet to be determined. This is an important issue given the species'proclivity for horizontal gene transfer, and the possibility that hypervirulent strains could be engineered by combining Burkholderia effectors. We imagine several applications resulting from experiments described below. First, a fundamental understanding of TsSSs and their associated effectors may allow more accurate prediction of the virulence characteristics of newly encountered uncharacterized isolates. Second, 1388 components may prove useful as immunogens and as diagnostic tools in antibody or antigen detection schemes. Third, numerous bioinformatics tools will be distributed to the scientific community. Finally, and most important, our efforts to better understand the TsSSs of B. pseudomallei will lead to a significantly improved understanding of virulence mechanisms and of bacterial pathogens'evolution. As is true for all the projects in our B. pseudomallei program, the experiments outlined below are highly collaborative and involve extensive intra- and inter-RCE collaborators.